Anti-PKMYT1 antibody [EPR27155-51] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PKMYT1 antibody [EPR27155-51] - BSA and Azide free (AB307147)

This data was developed using ab307146, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeABilized K-562 (human chronic myelogenous leukemia lymphoblast) cells lABelling PKMYT1 with ab307146 at 1/50 (10.42 ug/ml) dilution, followed by ab150081 Goat Anti-RABbit IgG H&L (Alexa Fluor? 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic staining in K-562 cell line.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor? 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-RABbit IgG H&L (Alexa Fluor? 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-PKMYT1 antibody [EPR27155-51] - BSA and Azide free (AB307147)

This data was developed using ab307146, the same antibody clone in a different buffer formulation.

PKMYT1 was immunoprecipitated from 0.35 mg K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug with ab307146 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307146 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug
Lane 2 : ab307146 IP in K562 whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307146 in K562 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 5 seconds

All lanes:

Immunoprecipitation - Anti-PKMYT1 antibody [EPR27155-51] (<a href='/en-us/products/primary-antibodies/pkmyt1-antibody-epr27155-51-ab307146'>ab307146</a>) at 1/1000 dilution

Lane 1:

K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 μg

Lane 2:

K562 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 60 kDa,70 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-PKMYT1 antibody [EPR27155-51] - BSA and Azide free (AB307147)

This data was developed using 307146, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : The molecular weight observed is consistent with what has been described in the literature (PMID : 9268380). Nocodazole treatment results in PKMYT1 phosphorylation and a shift of MW on the gel. Lanes 1-2 : 81 seconds, Lane 3 : 26 seconds. Exposure time :

All lanes:

Western blot - Anti-PKMYT1 antibody [EPR27155-51] (<a href='/en-us/products/primary-antibodies/pkmyt1-antibody-epr27155-51-ab307146'>ab307146</a>) at 1/1000 dilution

Lane 1:

Untreated K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 20 μg

Lane 2:

K562 treated with /mL nocodazole for 18 hours whole cell lysate 13 μg

Lane 3:

Untreated CCRF-CEM (human peripheral blood T lymphoblast) whole cell lysate 20 μg

Lane 4:

CCRF-CEM treated with /mL nocodazole for 18 hours whole cell lysate 20 μg

Lane 5:

K562 whole cell lysate 20 μg

Lane 6:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 20 μg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Observed band size: 60 kDa,70 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-PKMYT1 antibody [EPR27155-51] - BSA and Azide free (AB307147)

This data was developed using 307146, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 62 seconds Exposure time :

All lanes:

Western blot - Anti-PKMYT1 antibody [EPR27155-51] (<a href='/en-us/products/primary-antibodies/pkmyt1-antibody-epr27155-51-ab307146'>ab307146</a>) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) transfected with scrambled siRNA control whole cell lysate 20 μg

Lane 2:

K-562 transfected with siRNA specifically targeti PKMYT1 whole cell lysate 20 μg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 60 kDa,70 kDa

false

Exposure time: 62s

• WB

Lab

Western blot - Anti-PKMYT1 antibody [EPR27155-51] - BSA and Azide free (AB307147)

This data was developed using ab307146, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time :
Lane 1 : 60 seconds
Lane 2 : 73 seconds

MYT1 is a regulator during mitosis, it may shift to different migrating forms in mitotic lysates causing different observed band sizes. PMID : 23241949, PMID : 9268380)

All lanes:

Western blot - Anti-PKMYT1 antibody [EPR27155-51] (<a href='/en-us/products/primary-antibodies/pkmyt1-antibody-epr27155-51-ab307146'>ab307146</a>) at 1/1000 dilution

Lane 1:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate (batch 1) at 20 µg

Lane 2:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate (batch 2) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 55 kDa

Observed band size: 45-70 kDa

false